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- EMDB-1026: Microscopic evidence for a minus-end-directed power stroke in the... -
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Basic information
Entry | Database: EMDB / ID: EMD-1026 | |||||||||
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Title | Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd. | |||||||||
![]() | helical reconstruction of microtubule motor complex AMPPNP state | |||||||||
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Biological species | ![]() ![]() | |||||||||
Method | helical reconstruction / cryo EM / negative staining | |||||||||
![]() | Wendt TG | |||||||||
![]() | ![]() Title: Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd. Authors: Thomas G Wendt / Niels Volkmann / Georgios Skiniotis / Kenneth N Goldie / Jens Müller / Eckhard Mandelkow / Andreas Hoenger / ![]() Abstract: We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various ...We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncd's dimeric X-ray structure to generate a mechanistic model for force transduction. To visualize the alpha-helical coiled-coil neck better, we engineered an SH3 domain to the N-terminal end of our ncd construct (296-700). Ncd exhibits strikingly different nucleotide-dependent three-dimensional conformations and microtubule-binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X-ray structure with stable interactions between the neck and motor core domain. Minus-end-directed movement is based mainly on two key events: (i) the stable neck-core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus-end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus-end direction. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 602.2 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.3 KB 10.3 KB | Display Display | ![]() |
Images | ![]() | 35.3 KB | ||
Filedesc layerLines | ![]() | 12 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | helical reconstruction of microtubule motor complex AMPPNP state | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 5.53 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Ncd296 motor fragment from Drosophila Melanogaster AMPPNP state
Entire | Name: Ncd296 motor fragment from Drosophila Melanogaster AMPPNP state |
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Components |
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-Supramolecule #1000: Ncd296 motor fragment from Drosophila Melanogaster AMPPNP state
Supramolecule | Name: Ncd296 motor fragment from Drosophila Melanogaster AMPPNP state type: sample / ID: 1000 / Oligomeric state: dimer / Number unique components: 2 |
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-Macromolecule #1: Ncd296 dimer
Macromolecule | Name: Ncd296 dimer / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 92 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #2: tubulin dimer
Macromolecule | Name: tubulin dimer / type: protein_or_peptide / ID: 2 / Name.synonym: alpha beta tubulin Details: bovine tubulin was purchased from Cytoskeleton Inc. (Denver,CO) Oligomeric state: polymer of alpha beta dimers / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 100 KDa |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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![]() | helical reconstruction |
Aggregation state | filament |
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Sample preparation
Concentration | 1.9 mg/mL |
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Buffer | pH: 6.8 / Details: 80mM PIPES, 2mM MgCl2, 2mM AMPPNP |
Staining | Type: NEGATIVE / Details: plunge-frozen in liquid ethane |
Grid | Details: quantifoil holey grids |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER Details: Vitrification instrument: standard guillotine. vitrification carried out at room temperature |
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Electron microscopy
Microscope | FEI/PHILIPS CM200FEG/ST |
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Temperature | Average: 93 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected at 150K mag |
Details | low dose imaging |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 21 µm / Number real images: 14 / Od range: 0.9 / Bits/pixel: 8 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 160 kV / Electron source: TUNGSTEN HAIRPIN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.26 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 38000 |
Sample stage | Specimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN |
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Image processing
Details | 257 subunits per 137 helical turns |
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Final reconstruction | Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Software - Name: PHOELIX Details: final map was calculated from 25 datasets of near and far sides |